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Podophyllotoxin is an anticancer compound found in Indian mayapple (Podophyllum emodii Wall.), American mayapple (Podophyllum peltatum L.), and other species. Podophyllotoxin and its derivatives are used in several commercially available pharmaceutical products such as the anticancer drugs etoposide, teniposide, and etopophos. Currently, the commercial production of podophyllotoxin is based on Indian mayapple. The objective of this study was to estimate podophyllotoxin concentration in American mayapple across its natural habitats in the eastern United States and to identify high podophyllotoxin types that could be used for further selection and cultivar development. Analyses of American mayapple leaves collected from 37 mayapple colonies across 18 states indicated a significant variation in podophyllotoxin, α-peltatin, and β-peltatin content and the presence of chemotypes. Overall, the concentrations of podophyllotoxin, α-peltatin, and β-peltatin in the collected accessions ranged from below detectable levels to 45.1, 47.3, and 7.0 mg·g−1 dry weight, respectively. We classified American mayapple accessions into seven groups: 1) with very high concentration of podophyllotoxin (greater than 20 mg·g−1) and no α- or β-peltatin; 2) high podophyllotoxin (greater than 10 mg·g−1) and no α-peltatin but trace amounts of β−peltatin; 3) medium podophyllotoxin (1 to 10 mg·g−1) and no α- or β-peltatin; 4) low podophyllotoxin (0.05 to 1 mg·g−1) and high α-peltatin; 5) trace amounts of podophyllotoxin and high concentration of α-peltatin and α-peltatin; 6) high α-peltatin and trace amounts of podophyllotoxin or β−peltatin; and 7) high α−peltatin and no podophyllotoxin or β-peltatin. American mayapple was found to grow on various soil types with a range of pH (4.6 to 7.6) and dissimilar concentrations of phytoavailable soil nutrients. Tissue zinc concentration was positively correlated to podophyllotoxin, whereas soil and tissue phosphorus was positively correlated to the concentration of α-peltatin. The results from this study may contribute toward the development of high podophyllotoxin-containing varieties of American mayapple and the development of a new cash crop for American farmers.